Background: HSP90 protects the cells from heat stress and facilitates protein maturation and stability. The full genome sequences of piroplasms contain two putative HSP90 proteins, which are yet uncharacterized. To this end, the two putative HSP90 proteins of Babesia orientalis were identified and characterized by molecular and in silico methods. Methods: The two putative proteins in B. orientalis genome showing homology with putative HSP90 of other piroplasms were cloned and sequenced. A computational analysis was carried out to predict the antigenic determinants, structure and function of these proteins. The interactions of two HSP90 isoforms with respective inhibitors were also examined through docking analysis. Results: The length of BoHSP90-A gene (amplified from gDNA) was 2706 bp with one intron from position 997 to 1299 bp. This gene amplified from cDNA corresponded to full length CDS with an open reading frame (ORF) of 2403 bp encoding a 800 amino acid (AA) polypeptide with a predicted size of 91.02 kDa. The HSP90-B gene was intronless with an ORF of 2349 bp, and predicted polypeptide comprised of 797 AA with a size of 90.59 kDa. The AA sequences of these two proteins of B. orientalis were the most identical to those of B. bovis. The BoHSP90-A and BoHSP90-B were recognized as 90 kDa in the parasite lysate by the rabbit antisera raised against the recombinant BoHSP90 proteins. The anti-B. orientalis buffalo serum reacted with the rBoHSP90s expressed in E. coli, indicating that these proteins might be secreted by the parasite before entry into host cells. The overall structure and functional analyses showed several domains involved in ATPase activity, client protein binding and HSP90 dimerization. Likewise, several HSP90 inhibitors showed binding to ATP binding pockets of BoHSP90-A and BoHSP90-B, as observed through protein structure-ligand interaction analysis. Conclusions: The two putative HSP90 proteins in B. orientalis were recognized as 90 kDa. The rBoHSP90-A and rBoHSP90-B were reacted with the B. orientalis infected buffalo serum. The computational structure and functional analyses revealed that these two proteins may have chaperonic activity. The protein structure-ligand interaction analyses indicated that these two proteins had many drug target sites.